In vitro electroporation of the lower rhombic lip of midgestation mouse embryos.

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.5. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation. This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

Hindbrain rhombic lip is comprised of discrete progenitor cell populations allocated by Pax6.

The lower rhombic lip (LRL) is a germinal zone in the dorsal hindbrain productive of tangentially migrating neurons, streaming extramurally (mossy fiber neurons) or intramurally (climbing fiber neurons). Here we show that LRL territory, operationally defined by Wnt1 expression, is parceled into molecular subdomains predictive of cell fate. Progressing dorsoventrally, Lmx1a and Gdf7 expression identifies the primordium for hindbrain choroid plexus epithelial cells; Math1, for mossy fiber neurons; and immediately ventral to Math1 yet within Wnt1(+) territory, a climbing fiber primordium dominated by Ngn1-expressing cells. Elimination of Pax6 results in expansion of this Ngn1(+) progenitor pool and reduction in the Math1(+) pool, with accompanying later enlargement of the climbing fiber nucleus and reductions in mossy fiber nuclei. Pax6 loss also disrupts Msx expression cell-nonautonomously, suggesting Pax6 may influence LRL progenitor identity indirectly through potentiating BMP signaling. These studies suggest that underlying the diversity and proportions of fates produced by the LRL is a precise suborganization regulated by Pax6.

The role of E2F4 in adipogenesis is independent of its cell cycle regulatory activity.

The E2F and pocket protein families are known to play an important role in the regulation of both cellular proliferation and terminal differentiation. In this study, we have used compound E2F and pocket protein mutant mouse embryonic fibroblasts to dissect the role of these proteins in adipogenesis. This analysis shows that loss of E2F4 allows cells to undergo spontaneous differentiation. The ability of E2F4 to prevent adipogenesis seems to be quite distinct from the known properties of E2F. First, it can be separated from any change in either E2F-responsive gene expression or cell cycle regulation. Second, it is a specific property of E2F4, and not other E2Fs, and it occurs independently of E2F4's ability to interact with pocket proteins. In addition, E2F4 loss does not override the differentiation defect resulting from pRB loss even though it completely suppresses the proliferation defect of Rb(-/-) mouse embryonic fibroblasts. This finding definitively separates the known, positive role of pRB in adipogenesis from its cell cycle function and shows that this pocket protein is required to act downstream of E2F4 in the differentiation process.

E2Fs regulate adipocyte differentiation.

When preadipocytes reenter the cell cycle, PPAR gamma expression is induced, coincident with an increase in DNA synthesis, suggesting the involvement of the E2F family of cell cycle regulators. We show here that E2F1 induces PPAR gamma transcription during clonal expansion, whereas E2F4 represses PPARg amma expression during terminal adipocyte differentiation. Using a combination of in vivo experiments with knockout and chimeric animals and in vitro experiments, we demonstrate that the absence of E2F1 impairs, whereas depletion of E2F4 stimulates, adipogenesis. E2Fs hence represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPAR gamma expression. This underscores the complex role of the E2F protein family in the control of both cell proliferation and differentiation.